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WO2025106807A1 - Classification d'échantillons à l'aide d'un enrichissement de méthylation - Google Patents

Classification d'échantillons à l'aide d'un enrichissement de méthylation Download PDF

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WO2025106807A1
WO2025106807A1 PCT/US2024/056111 US2024056111W WO2025106807A1 WO 2025106807 A1 WO2025106807 A1 WO 2025106807A1 US 2024056111 W US2024056111 W US 2024056111W WO 2025106807 A1 WO2025106807 A1 WO 2025106807A1
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strand
adapter
nucleic acid
sample
seq
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Jingru YU
Wei Gu
Lauren AHMANN
Yvette Ysabel YAO
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Leland Stanford Junior University
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Leland Stanford Junior University
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C12N9/222—Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes
    • C12N9/226—Class 2 CAS enzyme complex, e.g. single CAS protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/11—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with 2-oxoglutarate as one donor, and incorporation of one atom each of oxygen into both donors (1.14.11)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/21—Endodeoxyribonucleases producing 5'-phosphomonoesters (3.1.21)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/04—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in cyclic amidines (3.5.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers

Definitions

  • the present disclosure relates to methods of analyzing nucleic acids in a sample.
  • methods of analyzing a sample involving enriching for nucleic acids containing a CpG site in a sample and assessing methylation status in enriched nucleic acids.
  • Embodiments of the present disclosure include methods of preparing nucleic acids for analysis.
  • a method of preparing nucleic acids for analysis comprising: a) ligating a first adapter to target double-stranded nucleic acids in a sample, wherein the first adapter comprises a primer binding strand containing a first primer binding site; b) adding a nuclease that cuts at a motif containing a CpG site, if present in the target double-stranded nucleic acids in the sample, thereby producing a subpopulation of nucleic acid fragments ligated to the first adapter, wherein at least some nucleic acid fragments in the subpopulation comprise a first end containing a cut site flanking sequence and a second end ligated to the primer binding strand of the first adapter; and c) adding a second adapter to the sample, wherein a portion of the second adapter comprising a second primer landing site ligates to the nu
  • the motif containing a CpG site comprises CCGG (SEQ ID NO: 1), TCGA (SEQ ID NO: 2), CGCG (SEQ ID NO: 3), CCGC (SEQ ID NO: 4), GCGC (SEQ ID NO: 5), or ACGT (SEQ ID NO: 6).
  • the nuclease cuts at a 5’ location to the CpG site in the motif. [0009] In some embodiments, the nuclease cut between the first and second residues of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. [0010] In some embodiments, the cut site flanking sequence comprises CGG, CGA, GCG, CGC, or CGT.
  • the nuclease cuts at a motif containing a CpG site in each strand of the double- stranded target nucleic acid, if present in the sample, such that the subpopulation of nucleic acid fragments ligated to the first adapter are substantially double-stranded.
  • the second end of a first strand of the substantially double- stranded nucleic acid fragment is ligated to the primer binding strand of the first adapter and the second end of a second strand of the substantially double-stranded nucleic acid fragment is ligated to the blocking strand of the first adapter.
  • FIGS. 1A-1B show a schematic of an exemplary FLEXseq design and workflow.
  • FIG. 3B, 3C, and 3D show the theoretical distance from the CCGG motifs for all CpGs covered by EPIC array, CCGG flanks, and cell type markers.
  • FIG. 6A-6C show classification of cancer cell line titrations using microarray-based ML classifiers.
  • FIG. 6A is a schematic of microarray-based ME classification.
  • FIG. 6B shows t-SNE dimensionality reduction of the 2,508 TCGA tumor and control references across 45 classes.
  • FIG. 6C shows /-SNE dimensionality reduction of tumor cell line titrations into immune cell DNA.
  • Cell lines are MCF-7 (BRCA), CL-40 (COAD), and DBTRG (GBM).
  • FIGS. 7A-7F show computational inference of methylation haplotypes.
  • FIG. 7A is a schematic for inferring methylation beta values through haplotypes between disparate datasets.
  • the left panel illustrates that individual CpG sites are matched one-to-one between array and sequencing data, leaving some CpGs unmatched and unused, while the right panel presents an approach that leverages haplotypes (from a human DNA methylation atlas of purified benign cell types) to synchronize CpG behavior across the genome, maximizing molecular depth and expanding CpG coverage.
  • FIG. 7B shows a theoretical example of inferring CpG methylation values through haplotypes.
  • FIG. 7C shows original beta values are highly concordant with the average beta values after intersecting with the haplotypes.
  • FIG. 7D shows FLEXseq intersected beta values are concordant with the EPIC array data. All benchmarks were performed using the K562 cell line.
  • FIG. 7E shows frequencies and percentages of all CpGs with 5x, lOx, 20x, and 30x coverage by FLEXseq.
  • FIG. 7F shows frequencies and percentages of CpG overlap between FLEXseq and tumor classifiers, with coverage >5x and >10x.
  • the red bar indicates increased CpGs after the intersection, and the blue bar indicates the original CpGs.
  • the dot indicates the percentage based on the right y-axis label.
  • FIG. 8 is an Illustrative flowchart of development of the random forest classifier. New classifiers were trained using CpG markers that overlap between FLEXseq and array reference datasets. The training samples were used to train the random forest model and conducted score calibration using a ridge regression model. Then the test samples were fit to the random forest model to get the predicted scores. The predicted scores were translated to meaningful class probabilities in the ridge regression.
  • FIG. 9 shows performance of the TCGA ML classifier (2,508 reference samples, with 41 subclasses).
  • FIG. 10 shows performance of the CNS ML classifier (2,801 reference samples, with 91 subclasses).
  • FIGS. 11A-11E show classification of body fluid and FFPE tissue samples.
  • FIG. 11A shows a summary of copy number analysis and ML classification using the TCGA and CNS ML classifiers of 79 non-CSF samples. ML classifications were categorized as ‘Matched’ (confirmation of diagnosis with classifier score >0.3), ‘Misleading profile’ (mismatched cancer with score >0.3), or ‘Indeterminate’ (positive tumors with score ⁇ 0.3 or predicted as control).
  • FIG. 11B shows sample sources.
  • FIG. 11C shows a classifier score distributions of FLEXseq samples predicted as tumors, including 25 non-CSF body fluids, 20 FFPE brain tissues, and six gDNA samples of tumor cell line titrations from the TCGA ML classifier.
  • FIG. 1 ID and FIG. 1 IE show cases with misleading ML classification (BF3090 and TF112) classified to the gold standard in the Z-SNE;
  • FIGS. 12A-12B show tumor classification using FLEXseq ML classifier.
  • FIG. 12A is a schematic of FLEXseq classification based only on FLEXseq references.
  • FIG. 12B shows classifier scores for the microarray- and FLEXseq-based classifier across 12 matching tumors and six control samples. The grey line in the box plot connects the same sample in both groups.
  • FIG. 12C shows t-SNE dimensionality reduction of 57 FLEXseq samples colored by pathological classes.
  • FIG. 12D shows t-SNE dimensionality reduction colored by sample types (FFPE or body fluid cfDNA).
  • FIGS. 13A-13D deconvolution of in silico titrations, physical DNA titrations, and plasma.
  • FIG. 13A shows cell type proportions deconvoluted from in silico mixtures using the WGBS references (WGBS reads intersected with CCGG flanks). CpG- (blue) and fragment-level deconvolution methods (red) are shown. Error bars indicate the SD at each titration level.
  • FIG. 13B shows deconvolution of gDNA of B cells, monocytes, neutrophils, and T cells titrated into mixtures of three immune cell types, respectively.
  • FIG. 13A shows cell type proportions deconvoluted from in silico mixtures using the WGBS references (WGBS reads intersected with CCGG flanks). CpG- (blue) and fragment-level deconvolution methods (red) are shown. Error bars indicate the SD at each titration level.
  • FIG. 13B shows deconvolution of gDNA of B cells,
  • FIG. 13C shows fragment-level deconvolution of BRCA (tumor cell-of-origin, breast luminal epithelium), COAD (tumor cell-of-origin, colon epithelium), and GBM (approximate tumor cell-of-origin lineage, neuron and oligodendrocyte) cell line DNA titrated into mixtures of the same four immune cell types in (a).
  • the dark blue line indicates deconvolution with references exclusive to the approximate tumor cell type and background immune cells.
  • the dark red line indicates deconvolution with a broader set of reference cell types encompassing common brain metastases.
  • FIG. 13D shows deconvolution of healthy plasma samples (WBGS2), WBGS2 intersected with CCGG flanks to simulate FLEXseq data, and FLEXseq from a healthy donor (P2).
  • FIG. 14A-14B show establishing deconvolution classifier based on deconvoluted cell type proportions.
  • FIG. 14A is a schematic of deconvolution classification.
  • FIG. 14B shows Z- scores of the cell-of-origin of three common CNS tumors: 20 LUADs (vs. 36 non-LUADs), 21 DLBCs (vs. 35 non-DLBCs), and six BRCAs (vs. 50 non-BRCAs).
  • the boxplot x-axis indicates the target (solid dots)/non-target tumors (solid triangles), and the x-axis in the scatter plots indicates the z-score rankings of the target and non-target tumors.
  • the black dashed lines are at z-scores of 2.
  • the ‘x’ indicates tumor samples where the cell type is not ranked first.
  • LU AD lung adenocarcinoma
  • DLBC B-cell lymphoma
  • BRCA breast carcinoma.
  • FIGS. 15A-15D show deconvolution of CSF samples.
  • FIG. 15B shows Z-scores of the cell-of-origin in less common CNS metastases and primary CNS tumors (labeled Brain) in 56 CSF tumor samples.
  • FIG. 15C shows Z-scores of all 20 deconvoluted cell types across 56 CSF tumor samples as normalized by the negative controls.
  • FIG. 15D shows Z-scores of all 20 cell types, but normalized against non-target tumors.
  • FIG. 16A-16E show a CSF case-control study.
  • FIG. 16A shows a summary of ML (random forest model), deconvolution, and composite classification and copy number analysis of 106 CSF cfDNA samples by FLEXseq. The triangle indicates CNA-positive results based on WGS. Five references used in the ML classifier were excluded due to high percentages of T cells.
  • FIG. 16B shows a schematic of the workflow using a representative CSF case (BF3741) that leads to copy number and classification analyses.
  • FIG. 1C shows the composite classification workflow integrates the copy number analysis (evaluating tumor presence and purity) with the ML and deconvolution classification.
  • FIG. 16D shows ROC curves for ML (blue dotted line), deconvolution (red dashed line), and composite (black solid line) classification for LU AD, DLBC, and BRCA.
  • the curves only include samples analyzed by all three classifiers, excluding 53 CNA-negatives and seven indeterminates from deconvolution.
  • FIG. 16E shows a confusion matrix for all 106 CSF samples based on the composite classification workflow in (c). # The case is classified with the pathological group in the t-SNE plot and misclassified in the random forest model. * One indeterminate case was not included under each tumor type.
  • FIG. 17A-17D shows CfDNA inputs of CSF and non-CSF body fluids.
  • FIG. 17c shows CfDNA concentrations calculated based on the cfDNA input titration curve in (a).
  • the 92 CSF, 10 FNA saline wash fluid, nine abdominal/peritoneal/ascitic fluid (ABDO), and 18 pleural fluid (PLEU) samples were shown, which were extracted at Stanford and met quality metrics (Ct ⁇ 14 and >30 million deduplicated paired-end reads).
  • the Y-axis is logged.
  • FIG. 19 shows the on-target rate of using the Taql-v2 nuclease (NEB, part number R0149) targeting the 'TCGA' motif rather than Mspl. Cutsmart buffer was used. The on-target rate was estimated by dividing reads starting with CGA or TGA by the total reads on the side cut by the nuclease. Each on-target read is guaranteed to contain CpG methylation data based on the first position.
  • the numbers 7 and 8 arc contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.
  • amino acid refers to natural amino acids, unnatural amino acids, and amino acid analogs, all in their D and L stereoisomers, unless otherwise indicated, if their structures allow such stereoisomeric forms.
  • complementary and complementarity refer to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick base-paring or other non-traditional types of pairing.
  • the degree of complementarity between two nucleic acid sequences can be indicated by the percentage of nucleotides in a nucleic acid sequence which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 50%, 60%, 70%, 80%, 90%, and 100% complementary).
  • Exemplary moderate stringency conditions include overnight incubation at 37° C in a solution comprising 20% formamide, 5xSSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5xDenhardt’s solution, 10% dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed by washing the filters in IxSSC at about 37-50° C., or substantially similar conditions, e.g., the moderately stringent conditions described in Sambrook et al., infra.
  • High stringency conditions are conditions that use, for example (1) low ionic strength and high temperature for washing, such as 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate (SDS) at 50° C, (2) employ a denaturing agent during hybridization, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin (BSA)/0.1% Ficoll/0.1% polyvinylpyrrolidone (PVP)/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride and 75 mM sodium citrate at 42° C., or (3) employ 50% formamide, 5xSSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1 % sodium pyrophosphate, 5xDenhardt’s solution, sonicated salmon sperm DNA (50 pg/ml), 0.1% SDS, and 10% dextran sul
  • nucleic acid or a “nucleic acid sequence” refers to a polymer or oligomer of pyrimidine and/or purine bases, preferably cytosine (C), thymine (T), and uracil (U), and adenine (A) and guanine (G), respectively.
  • C cytosine
  • T thymine
  • U uracil
  • G adenine
  • G guanine
  • the present technology contemplates any deoxyribonucleotide, ribonucleotide, or peptide nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated, or glycosylated forms of these bases, and the like.
  • LNA locked nucleic acid
  • cyclohexenyl nucleic acids see Wang, J. Am. Chem. Soc., 122: 8595-8602 (2000), incorporated herein by reference
  • ribozyme a ribozyme
  • nucleic acid or “nucleic acid sequence” may also encompass a chain comprising non-natural nucleotides, modified nucleotides, and/or non- nucleotide building blocks that can exhibit the same function as natural nucleotides (i.e., “nucleotide analogs”); further, the term “nucleic acid sequence” as used herein refers to an oligonucleotide, nucleotide or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin, which may be single or double-stranded, and represent the sense or antisense strand.
  • nucleic acid refers to a polymeric form of nucleotides of any length, cither dcoxyribonuclcotidcs or ribonucleotides, or analogs thereof.
  • a “peptide” or “polypeptide” is a linked sequence of two or more amino acids linked by peptide bonds.
  • the peptide or polypeptide can be natural, synthetic, or a modification or combination of natural and synthetic.
  • Polypeptides include proteins such as binding proteins, receptors, and antibodies. The proteins may be modified by the addition of sugars, lipids or other moieties not included in the amino acid chain.
  • the terms “polypeptide” and “protein,” are used interchangeably herein.
  • percent sequence identity refers to the percentage of nucleotides or nucleotide analogs in a nucleic acid sequence, or amino acids in an amino acid sequence, that is identical with the corresponding nucleotides or amino acids in a reference sequence after aligning the two sequences and introducing gaps, if necessary, to achieve the maximum percent identity.
  • additional nucleotides in the nucleic acid, that do not align with the reference sequence are not taken into account for determining sequence identity.
  • Methods and computer programs for alignment are well known in the art, including BLAST, Align 2, and FASTA.
  • provided herein are methods. In some embodiments, provided herein are methods of preparing nucleic acids for analysis. In some embodiments, the methods provide herein are used to prepare nucleic acids in a sample for analyses such as amplification (e.g. PCR), sequencing, or methylation analyses. In some embodiments, provided herein are methods of classifying methylation status of a sample.
  • the methods provided herein involve contacting a sample with a first adapter that ligates to target nucleic acids in the sample, cutting the adapter-ligated nucleic acids with a nuclease that cuts at a motif containing a CpG site, if present, and adding a second adapter to the sample that ligates (e.g. at the cute site) to the subpopulation of adapted-ligated nucleic acid fragments resulting from cutting. Accordingly, the methods provided herein enrich for nucleic acids cut at a motif containing a CpG site, which provide valuable information from the cute site flanking region regarding methylation status of a sample.
  • the motif containing a CpG site comprises CCGG (SEQ ID NO: 1), TCGA (SEQ ID NO: 2), CGCG (SEQ ID NO: 3), CCGC (SEQ ID NO: 4), GCGC (SEQ ID NO: 5), or ACGT (SEQ ID NO: 6).
  • the nuclease cuts at a 5’ location to the CpG site in the motif.
  • the nuclease cutting at a 5’ location to the CpG site in the motif produces a sequence referred to herein as a “flanking sequence” or a “cut site flanking sequence”, which contains the CpG site.
  • the cut site flanking sequence comprises CGG, CGA, GCG, CGC, or CGT.
  • the methods described herein produce nucleic acids containing a cut site flanking sequence covalently bonded at one end to a portion of the first adapter and covalently bonded at a second end to a portion of the second adapter.
  • the first adapter and the second adapter each comprise a primer landing site, which facilitates amplification and/or sequencing of the nucleic acids.
  • the methods comprise performing a conversion step to selectively convert cytosine residues in the sample depending on their methylation status to a different nucleic acid besides cytosine, and generation a methylation profile for the sample by sequencing.
  • the methods comprise sequencing the nucleic acids containing the cut site flanking sequence covalently bonded to the first adapter and to the second adapter by nanopore sequencing.
  • nanopore sequencing no enzymatic conversion step to selectively convert cytosine residues in the sample depending on their methylation status is needed.
  • the adapters need not contain primer landing sites.
  • the first adapter ligated to the target double- stranded nucleic acid in the sample and/or the second adapter ligated to the population of nucleic acid fragments e.g.
  • nuclease that cuts at a motif containing a CpG site in the target double-stranded nucleic acids in the sample do not contain a primer landing site and instead contain a motor protein which helps to pass a given nucleic acid through a given nanopore of a nanopore sequencing device/system.
  • the method comprises ligating a first adapter to target doublestranded nucleic acids in a sample.
  • the first adapter comprises a primer binding strand containing a first primer landing site.
  • the primer binding strand of the first adapter ligates to a first strand of the target nucleic acids in the sample.
  • This first strand of the target double-stranded nucleic acid in the sample is subsequently cut by a nuclease that cuts at the motif containing a CpG site, if present in the first strand, thereby producing a nucleic acid fragment.
  • This nucleic acid fragment is subsequently ligated to a second adapter containing a second primer landing site, and is therefore ultimately able to be substantially amplified due to the presence of both the first primer landing site and the second primer landing site.
  • a second strand of the target double- stranded nucleic acids in the e.g. the strand complementary to the first strand
  • the methods provided herein facilitate selective substantial amplification and sequencing of a first strand, but not a second strand, of target double- stranded nucleic acids in a sample.
  • the second strand of the target double-stranded nucleic acids is ligated to a blocking strand of the first adapter, thereby ultimately preventing substantial amplification of the second strand as described in more detail below.
  • the second strand of the target nucleic acids does not ligate to the first adapter, and is therefore not able to be substantially amplified due to the absence of at least a first primer landing site.
  • the double-stranded end portion is a blunt end (e.g. lacks an overhang).
  • the 5’ end of the adapter contains the first primer landing site.
  • a 5’ end of the primer binding strand ligates to a 3’ end of a first strand of the target double-stranded nucleic acid.
  • the first adapter comprises a primer binding strand.
  • the double stranded end portion comprises a primer binding strand.
  • the term “primer binding strand” indicates that the strand contains a primer landing site and thus facilitates downstream amplification of the nucleic acid containing the primer landing site in the presence of the primer.
  • the landing site may be designed to facilitate direct or indirect primer binding.
  • the landing site facilitates direct primer binding.
  • Direct primer binding indicates that a first primer is added to the sample and directly binds to the primer landing site on the primer binding strand of the first adapter.
  • the primer landing site of the first adapter is complementary to the first primer.
  • the landing site facilitates indirect primer binding. Indirect primer binding indicates that a second primer added to the sample binds to second (e.g. separate) primer landing site (e.g., on the second adapter) and during subsequent extension of the primer-bound nucleic acid, the primer landing site for a first primer is created.
  • the primer landing site of the first adapter is the reverse complement of the primer sequence for the first primer.
  • the landing site if it has cytosines, have nucleic acid modifications that will resist conversion to another nucleic acid upon methylation conversion.
  • the double stranded end portion further comprises a blocking strand.
  • blocking strand indicates that the strand contains features which prevent substantial amplification of the strand or lacks essential features for substantial amplification of the strand (e.g. during PCR, during sequencing). Accordingly, the blocking strand is not substantially amplified in downstream PCR or sequencing steps.
  • the mechanism by which the blocking strand is not substantially amplified or sequenced can be active (e.g. the blocking strand may contain a moiety that prevents substantial amplification and/or sequencing) or passive (e.g. the blocking strand may lack a moiety that would otherwise facilitate amplification and/or sequencing).
  • a blocking strand contains a moiety to prevents ligation of the blocking strand to the target nucleic acid yet allows for the ligation of the primer landing strand to the target nucleic acid.
  • a blocking strand contains a moiety that prevents attachment of primers to the blocking strand for subsequent amplification and/or sequencing steps.
  • a blocking strand contains a moiety that interferes with the ability of a polymerase during amplification to transverse across the molecule.
  • a blocking strand contains a moiety that allows the strand to be disconnected.
  • a blocking strand contains a moiety allows for at least a portion of the blocking strand to be degraded.
  • a blocking strand lacks a primer landing site (e.g. lacks a direct primer landing site and/or lacks an indirect primer landing site).
  • the blocking strand comprises a nucleotide that is modified during a conversion step such that primer landing to the blocking strand is disrupted upon conversion.
  • a single-stranded ligation of the primer binding strand of the first adapter to a target double stranded nucleic acid occurs.
  • the primer binding strand of the first adapter ligates to a first strand of the target double- stranded nucleic acid, and the second strand of the target nucleic acid does not ligate to the first adapter.
  • the first adapter comprises a primer binding strand and does not comprise a blocking strand.
  • the first adapter is substantially double-stranded in its entirety.
  • the first adapter is a Y-shaped or a U-shaped adapter.
  • the first adapter is a Y-shaped or a U-shaped adapter comprising a substantially double-stranded end containing the primer binding strand and the blocking strand, and a singlestranded portion at the non-ligating end of the adapter.
  • a Y-shaped or U- shaped adapter has non-complementary (therefore non-double stranded) strands at the nonligating end of the adapter.
  • the primer binding strand of the first adapter may comprise any suitable sequence.
  • the primer binding strand of the first adapter comprises a first primer landing site having a sequence complementary to a first primer sequence
  • the second primer landing site e.g. of the second adapter
  • the sequences of the first primer, first primer landing site, second primer and the second primer landing site may vary so long as sufficient complementarity between the first primer and the first primer landing site, and the second primer and the second primary landing site, is achieved for exponential amplification.
  • the primer binding strand is not originally present, but through methylation conversion, can be created by swapping nucleic acids (e.g. cytosine for uracil).
  • the original sequence that becomes the primer binding strand is also regarded indirectly as a form of the primer binding strand.
  • the primer binding strand of the first adapter comprises the sequence 5’GATCGGAAGAGCGTCGT-3’. (SEQ ID NO: 7). In some embodiments, the primer binding strand of the first adapter comprises a sequence having at least 88% sequence identity with SEQ ID NO: 7.
  • the blocking strand of the first adapter is substantially complementary to (e.g. is substantially the reverse complement of) the primer binding strand of the first adapter, thus facilitating the formation of the double- stranded end portion of the adapter. In some embodiments, the blocking strand of the first adapter comprises the sequence 5’- ACGCTCTTCCGATCT -3’ (SEQ ID NO: 8).
  • the blocking strand of the first adapter comprises a sequence having at least 88% sequence identity with SEQ ID NO: 8.
  • the primer binding strand of the first adapter comprises a sequence having at least 88% sequence identity with SEQ ID NO: 7 and the blocking strand of the first adapter comprises a sequence having at least 88% sequence identity with SEQ ID NO: 8.
  • the primer binding strand of the first adapter comprises the sequence of SEQ ID NO: 7 and the blocking strand of the first adapter comprises the sequence of SEQ ID NO: 8. [0077]
  • the primer binding strand of the first adapter comprises the sequence 5’-GATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT-3’ (SEQ ID NO: 9).
  • the primer binding strand of the first adapter comprises a sequence having at least 80% sequence identity (e.g. at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with SEQ ID NO: 9.
  • the cytosines (C) in the primer binding strand sequence are resistant to methylation conversion (e.g. they are methylated).
  • the blocking strand of the first adapter comprises the sequence 5’- ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’ (SEQ ID NO: 10).
  • the blocking strand of the first adapter comprises a sequence having at least 80% sequence identity (e.g. at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with SEQ ID NO: 10.
  • the primer binding strand of the first adapter comprises a sequence having at least 80% sequence identity (e.g.
  • the blocking strand of the first adapter comprises a sequence having at least 80% sequence identity (e.g. at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with SEQ ID NO: 10.
  • the primer binding strand comprises the sequence of SEQ ID NO: 9 and the blocking strand comprises the sequence of SEQ ID NO: 10.
  • the 5’ end of the primer binding strand of the first adapter covalently attaches to the 3’ end of a target nucleic acid in the sample.
  • the 5’ end of the primer binding strand of the first adapter comprises a moiety that facilitates covalent attachment of the primer binding strand to target DNA sequences during ligation.
  • the 5’ end of the primer binding sequence comprises a phosphate.
  • a 5’ phosphate is not added to the sample prior to ligation to prevent covalent attachment of the first adapter’ s blocking strand to the target nucleic acids in the sample.
  • 5' phosphates are removed from the sample prior to ligation to prevent covalent attachment of the first adapter’ s blocking strand to the target nucleic acids in the sample.
  • the first adapter sequence is partially modified such that the cytosine residues present in at least the primer binding strand are not converted to other residues during subsequent conversion (e.g. enzymatic conversion, bisulfite conversion) steps.
  • the cytosine residues in the primer binding strand are methylated or hydroxymethylated to resist conversion (e.g. to uracil, to thymine) upon addition of a converting enzyme. Whether the residues are methylated or hydroxymethylated depends on the specific enzyme(s) to be added during a subsequent conversion step.
  • the blocking strand lacks a moiety (e.g. a phosphate group) that would otherwise facilitate covalent attachment of the blocking strand to a target nucleic acid in the sample.
  • a moiety e.g. a phosphate group
  • the first adapter sequence is partially modified such that the cytosine residues present in at least the primer binding strand are not converted to other residues during subsequent conversion (e.g. enzymatic conversion, bisulfite conversion) steps.
  • the cytosine residues in the primer binding strand are methylated or hydroxymethylated to resist conversion (e.g. to uracil, to thymine) upon addition of a converting enzyme or chemical. Whether the residues are changed due to its methylated or hydroxymethylated status depends on the specific enzyme(s) to be added during a subsequent conversion step.
  • cytosine residues in the blocking strand are not modified (e.g.
  • a hemimethylated adapter refers to an adapter wherein cytosine residues in the binding strand are methylated or hydroxymethylated, and cytosine residues in the blocking strand are not.
  • the primer binding strand ligates (e.g. covalently) to a strand of the double- stranded target nucleic acid in the sample.
  • the 5’ portion of the primer binding strand ligates to a 3’ end of the target nucleic acid.
  • the primer binding strand and the blocking strand each ligate a strand of the double-stranded target nucleic acid in the sample.
  • the primer binding strand of the first adapter ligates to a first strand of the double-stranded target nucleic acid
  • the blocking strand of the first adapter ligates to a second strand of the double- stranded target nucleic acid in the sample.
  • a population of substantially double-stranded target nucleic acids are produced wherein one strand is bound to the primer binding strand and the other strand is bound to the blocking strand.
  • the primer binding strand binds to a strand of the double-stranded target nucleic acid in the sample, and the blocking strand does not bind to target nucleic acid.
  • the primer binding strand binds to a strand of the doublestranded target nucleic acid in the sample, and the other strand of the double- stranded target nucleic acid does not bind to either the primer binding strand or the blocking strand of the first adapter and is thus ultimately not substantially amplified or sequenced due to lacking the first primer landing site.
  • the first adapter comprises a primer binding strand that binds to a first strand of the double- stranded target nucleic acid in the sample, and the second strand of the double-stranded target nucleic acid does not bind to the first adapter and is thus ultimately not substantially amplified or sequenced.
  • the method comprises adding a nuclease that cuts at a motif containing a CpG site, if present in the target nucleic acids in the sample.
  • the motif containing a CpG site comprises CCGG (SEQ ID NO: 1), TCGA (SEQ ID NO: 2), CGCG (SEQ ID NO: 3), CCGC (SEQ ID NO: 4), GCGC (SEQ ID NO: 5), or ACGT (SEQ ID NO: 6).
  • the nuclease cuts at a 5’ location to the CpG site in the motif.
  • the nuclease cuts at a 5’ location to the CpG site in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.
  • the cute site flanking sequence contains the CpG site.
  • the cut site flanking sequence comprises CGG, CGA, GCG, CGC, or CGT.
  • the motif containing a CpG site comprises CCGG (SEQ ID NO: 1).
  • the nuclease cuts between the first C and second C in the CCGG sequence. Accordingly, in such embodiments the nuclease produces a first nucleic acid fragment wherein the CCGG cut site flanking sequence begins with CGG.
  • nuclease Any suitable nuclease may be used to cut at the motif containing a CpG site.
  • the nuclease is MspI, Taql-v2, AccII, Acil, AspLEI, HPall, HpyCH4IV.
  • the nuclease is a type II nuclease.
  • the nuclease has impaired cleavage when the CpG site at the motif is methylated.
  • the nuclease is MspI and the motif containing a CpG site comprises CCGG (SEQ ID NO: 1).
  • the nuclease is Hpall and the motif containing a CpG site comprises CCGG (SEQ ID NO: 1). In some embodiments the nuclease is Taql-v2 and the motif containing a CpG site comprises TCGA (SEQ ID NO: 2). In some embodiments, the nuclease is AccII and the motif containing a CpG site comprises CGCG (SEQ ID NO: 3). In some embodiments the nuclease is Acil and the motif containing a CpG site comprises CCGC (SEQ ID NO: 4). In some embodiments, the nuclease is AspLEI and the motif containing a CpG site comprises GCGC (SEQ ID NO: 5). In some embodiments, the nuclease is HpyCH4IV and the motif containing a CpG site comprises ACGT (SEQ ID NO: 6).
  • the nuclease is a CRISPR/Cas nuclease (e.g. CRISPR/Cas9, CRISPR/Casl2, CRISPR/Cas 13, etc.).
  • the nuclease is a CRISPR/nuclease highly specific for targeted sites.
  • CRISPR-Cas9 nuclease conveniently requires only a subset of the CCGG motif — the ‘GG’ — on the PAM site to make a cut.
  • CRISPR/nuclease targets a plurality of sites, including ones that contain CCGG. Accordingly, Cas9 targeting is compatible with the methods described herein.
  • residual phosphates are removed using a suitable phosphatase prior to adding the nuclease to the sample.
  • the method incubates the sample with a suitable phosphatase to remove residual phosphates and subsequently adding the nuclease to the sample.
  • Cutting at the motif containing a CpG site produces a subpopulation of nucleic acid fragments ligated to the first adapter.
  • at least some nucleic acid fragments in the subpopulation comprise a first end containing a cut site flanking sequence and a second end ligated to the primer landing strand of the first adapter.
  • some nucleic acid fragments in the subpopulation comprise a first end containing a cut site flanking sequence and a second end ligated to the primer landing strand of the first adapter, and some nucleic acid fragments comprise a first end containing a cut site flanking sequence and a second end ligated to the blocking strand of the first adapter.
  • the target double-stranded nucleic acid comprises a first strand that ligates to the primer binding strand of the first adapter and a second strand that ligates to the blocking strand of the first adapter.
  • the first strand produces a fragment comprising a first end containing a cut site flanking sequence and a second end ligated to the primer landing strand of the first adapter
  • the second strand produces a fragment comprising a first end containing a cut site flanking sequence and a second end ligated to the blocking strand of the first adapter
  • one end of a first strand in the substantially double- stranded nucleic acid fragment is ligated to the primer binding strand of the first adapter and one end of a second strand in the substantially double- stranded nucleic acid fragment is ligated to the blocking strand of the first adapter.
  • one end of a first strand in the substantially double- stranded nucleic acid fragment is ligated to the primer binding strand of the first adapter and one end of a second strand in the substantially double- stranded nucleic acid fragment is not ligated to the first adapter.
  • the method further comprises adding a second adapter to the sample.
  • the second adapter is designed to selectively ligate to nucleic acid fragments generated as a result of cleavage of target nucleic acids at motif containing a CpG site. Accordingly, the second adapter does not substantially ligate to uncleaved nucleic acids (e.g. nucleic acids in the sample lacking a motif containing a CpG site). Therefore, the methods provided herein produce a population of nucleic acid that do not contain a motif containing a CpG site and arc therefore ligated to the first adapter, but not ligated to the second adapter. These nucleic acids are not able to be substantially amplified, due to the absence of the second primer landing site, as described in more detail below.
  • the second adapter contains a second primer landing site.
  • a portion of the second adapter comprising the second primer landing site selectively ligates to the nucleic acid fragments ligated the first adapter.
  • Ligation of the second adapter to the nucleic acid fragments ligated to the first adapter forms a population of nucleic acid strands that are ligated (e.g. covalently bound) to both the first adapter and the second adapter.
  • ligation of the second adapter to the nucleic acid fragments ligated to the first adapter produces a population of nucleic acid strands wherein one end of the strand is ligated (e.g.
  • ligation of the second adapter to the nucleic acid fragments ligated to the first adapter produces a population of nucleic acid strands comprising the first primer landing site, the cut site flanking sequence, and the second primer landing site.
  • ligation of the second adapter to the nucleic acid fragments ligated to the first adapter produces a population of amplifiable nucleic acid strands (e.g.
  • strands wherein one end of the strand is ligated to the primer binding strand of the first adapter and the other end of the strand is ligated to a portion of the second adapter comprising the second primer landing site, as described above) and a population of non-amplifiable nucleic acid strands wherein one end of the strand is not ligated to either strand of the first adapter and the other end of the strand is ligated to a portion of the second adapter comprising the second primer landing site.
  • the second adapter is double- stranded and is intended for double stranded ligation. In some embodiments, the adapter is intended for single stranded ligation. In some embodiments, the portion of the second adapter comprising a second primer landing site comprises the sequence 5’-GACTGGAGTTCAGACGTGTGCTCT TCCGATCT-3’. (SEQ ID NO: 12). In some embodiments, the portion of the second adapter comprising a second primer landing site comprises a sequence having at least 80% sequence identity (e.g.
  • the second adapter is a single-stranded adapter comprising a sequence having at least 80% sequence identity (e.g. at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with SEQ ID NO: 12.
  • the second adapter is a single-stranded adapter comprising a sequence having at least 80% sequence identity (e.g. at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with SEQ ID NO: 12.
  • the cytosines (C) in the sequence are resistant to methylation conversion (e.g. they are methylated).
  • the second adapter is a double- stranded adapter, wherein a first strand of the adapter comprises a sequence having at least 80% sequence identity (e.g. at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) with SEQ ID NO: 12 and the second strand is substantially complementary to (e.g. is substantially the reverse complement of) the sequence of the first strand.
  • the second adapter is a double- stranded adapter wherein the first strand of the adapter comprises a sequence having at least 80% sequence identity with SEQ ID NO: 12 and the second strand of the adapter comprises a sequence having at least 80% sequence identity with 5’-GATCGGAAGAGCACACGTCTGAACTCCAGTC-3’ (SEQ ID NO: 13).
  • the first adapter and/or the second adapter does not comprise a primer landing site.
  • the methods herein involve nanopore sequencing to determine the sequence of a nucleic acid strand containing a cut site flanking sequence ligated at one end to the first adapter and ligated at the second end to the second adapter.
  • the first adapter and/or the second adapter need not contain a primer landing site, as no PCR amplification will be performed prior to nanopore sequencing.
  • a motor protein may be utilized in the first adapter and/or in the second adapter. Such a motor protein guides a given nucleic acid strand through the nanopore, facilitating sequencing of the strand.
  • the method comprises ligating a first adapter to target double- stranded nucleic acids in the sample, wherein the first adapter is a nanopore sequencing adapter, thereby producing a population of nucleic acid strands ligated to the first adapter.
  • the method comprises adding a nuclease that cuts at a motif containing a CpG site, if present in the target double-stranded nucleic acids in the sample, thereby producing a subpopulation of nucleic acid fragments comprising a cut site flanking sequence and at least one end ligated to the first adapter.
  • the method further comprises adding a second adapter to the sample that ligates to the nucleic acid fragments ligated to the first adapter, thereby producing nucleic acid strands comprising the cut site flanking sequence ligated at one end to the first adapter and ligated at the other end to the second adapter.
  • the first and/or second adapters may comprise motor proteins that guide the nucleic acid strands through a nanopore of a nanopore sequencing device.
  • a nanopore is able to determine sequencing information about the strand, including methylation status. Accordingly, such embodiments need not include primer landing sites on the adapters, as no PCR amplification is necessary prior to nanopore sequencing.
  • no enzymatic conversion step to selectively convert residues is needed prior to nanopore sequencing.
  • the cut site flanking sequence results from the nuclease cutting a single motif containing a CpG site in a target nucleic acid.
  • the nuclease cuts a single motif containing a CpG site in the target nucleic acid, which produces a cut site flanking sequence only in the 5’ or the 3’ direction from the cut site. This is in contrast to methods (e.g. RRBS) wherein only target nucleic acid with two cut sites are produced by a nuclease, producing a flanking sequenced sandwiched between the two cut sites.
  • RRBS e.g. RRBS
  • the second adapter contains the second primer landing site and the first primer landing site on the opposite strand and can accommodate target nucleic acid with two cut sites in addition to target nucleic acid with one cut site.
  • the cut site flanking sequence may be any suitable number of nucleotides in length. In some embodiments, the cut site flanking sequence is 10-2000 bases in length. In some embodiments, the cut site flanking sequence comprises is at least 20 bases in length. In some embodiments, the cut site flanking sequence is 20 to 300 bases in length (e.g. about 20 to 300 bases, about 25 to 250 bases, about 30 to 200 bases, about 35 to about 175 bases, about 40 to about 150 bases, or about 50 to about 100 bases. In some embodiments, the cut site flanking sequence is about 50 base pairs in length. In some embodiments, the cut site flanking sequence is about 70 to about 150 base pairs in length. The results presented herein demonstrate that for all CCGG flanking sequence lengths (e.g.
  • the methods provided herein further performing a conversion step to selectively convert cytosine residues in the sample depending on their methylation status to a different nucleic acid besides cytosine.
  • the conversion step may be performed in between adding the nuclease and adding the second adapter to the sample, or may be performed after adding the nuclease and adding the second adapter to the sample.
  • the conversion step may comprise bisulfite treatment or an enzymatic treatment. Cytosine methylation (5-methylcytosine, 5mC) and hydroxymethylation (5-hydroxylmethylcytosine, 5hmC) arc the most common epigenetic marks in the eukaryotic genome and can be used to evaluate methylation status of a sample.
  • unmodified cytosine residues arc converted.
  • methylated cytosine (5mC) residues and/or hydroxymethylated (5hmC) residues are converted.
  • the desired cytosine residues are converted to uracil or another base that is detectably dissimilar to cytosine. Exemplary methods for enzymatic conversion are described in PMID 34140313, 30804537, 37322153.
  • the conversion step selectively converts unmethylated cytosine residues in the sample to uracil residues.
  • bisulfite conversion can be used, wherein a sample is treated with bisulfite to selectively convert unmethylated, but not methylated or hydroxymethylated, cytosine bases to uracil. Exemplary methods for bisulfite conversion are described in (Frommer et al., 1992, Proc Natl Acad Sci USA 89:1827-31; Olek, 1996, Nucleic Acids Res 24:5064-6; EP 1394172).
  • methylated cytosine (5mC) residues and/or hydroxymethylated (5hmC) residues arc converted using an enzymatic treatment.
  • the enzymatic treatment may be performed using one enzyme or multiple enzymes.
  • the conversion step is an enzymatic conversion utilizing a ten-eleven translocation (TET)- related enzyme.
  • TET methylcytosine dioxygenases comprise three enzymes that catalyze the hydroxylation of DNA methyl cytosine (5mC) into 5-hydroxymethylcytosine (5hmC) and then further oxidize 5hmC to form 5-formylcytosine (5fC) and 5-carboxycytosine (5cAC).
  • oxidation products can be further reduced or converted into a suitable base detectably dissimilar from cytosine.
  • An exemplary method involving use of a TET-related enzyme is described in Liu, Y., Siejka-Zieliriska, P., Velikova, G. et al. Bisulfite-free direct detection of 5- methylcytosine and 5-hydroxymethylcytosine at base resolution. Nat Biotechnol 37, 424-429 (2019). https://doi.org/10.1038/s41587-019-0041-2.
  • the method involves contacting a sample with TET enzymes such that 5mC and 5hmC are oxidised to 5-carboxylcytosine (5caC) and subsequently reduced to dihydrouracil (DHU) by pyridine borane. DHU is then amplified and sequenced as thymine (T) during final sequencing.
  • TET enzymes such that 5mC and 5hmC are oxidised to 5-carboxylcytosine (5caC) and subsequently reduced to dihydrouracil (DHU) by pyridine borane.
  • DHU is then amplified and sequenced as thymine (T) during final sequencing.
  • the conversion step is an enzymatic conversion using an apolipoprotein B mRNA-editing catalytic polypeptide- like (APOBEC) enzyme.
  • APOBEC enzymes readily deaminate unmodified cytosine and 5mC, but not cytosines with other modifications.
  • One drawback of this approach is that it can only be used for measuring 5hmC, and does not distinguish 5mC from unmodified cytosine.
  • APOBEC enzymes can be used in conjunction with other suitable enzymatic modifications to facilitate investigation of both 5mC and 5hmC, as opposed to 5hmC alone.
  • the commercially available NEBNext Enzymatic Methyl-seq Kit uses TET2 to first oxidize 5mC and 5hmC, which provides protection of these bases during the subsequent addition of APOBEC.
  • APOBEC is then applied to deaminate the unmodified cytosines to uracils.
  • the modified bases 5rnC and 5hmC can be detected without harsh chemical treatments such as bisulfite.
  • the method further comprising selectively amplifying and/or sequencing the nucleic acid strands comprising the first adapter, the cut site flanking sequence, and the second adapter.
  • the method comprises selectively sequencing, by nanopore sequencing, the nucleic acid strands comprising the first adapter, the cut site flanking sequence, and the second adapter (as described above, the first adapter and/or the second adapter need not contain primer landing sites but instead may contain motor protein(s) that assist in nanopore sequencing.
  • the method comprises selectively amplifying and/or sequencing the nucleic acid strands comprising the first primer landing site, the cut site flanking sequence, and the second primer landing site (also referred to herein as the “amplifiable strands”).
  • amplification is performed by polymerase chain reaction (PCR) using amplification primers.
  • PCR polymerase chain reaction
  • amplification is performed using a first primer containing a sequence that is complementary to the primer landing site or the reverse complement thereof in the first adapter, and using a second primer containing a sequence that is complementary to the second primer landing site or the reverse complement thereof in the second adapter.
  • the sequence of the primers can vary depending on the sequence of the primer landing sites present within the first and second adapters.
  • the primer landing sites can be designed such that direct primer binding occurs (e.g. the primer is complementary to the sequence of the primer landing site and thus directly binds to the primer landing site) or indirect primer binding occurs (e.g. the primer is complementary to the reverse complement of the primer landing site generated during synthesis of the reverse complementary strand).
  • amplification is performed using a first primer (P5) comprising the sequence 5’-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’ (SEQ ID NO: 10).
  • a primer comprising the sequence of SEQ TD NO: 10 directly binds to the primer landing site of the primer binding strand of the first adapter.
  • the first primer comprises the sequence AATGATACGGCGACCACCGAGATCTACACCGAATACGACACTCTTTCCCTACACGAC GCTCTTCCGATCT (SEQ ID NO: 11).
  • amplification is performed using a second primer (P7) comprising the sequence GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT (SEQ ID NO: 14).
  • a primer comprising the sequence of SEQ ID NO: 14 binds to the reverse complement of second primer landing site (e.g. formed after the reverse complement of the second adapter is made).
  • the second primer comprises the sequence CAAGCAGAAGACGGCATACGAGATGTCGGTAAGTGACTGGAGTTCAGACGTGTGCT CTTCCGATCT (SEQ ID NO: 15).
  • the method further comprises sequencing the amplified nucleic acids.
  • sequencing provides valuable information about the methylation status of cut site flanking sequences obtained by such a method.
  • a variety of suitable sequencing methods and technologies may be used to determine the sequence of the nucleic acid strands.
  • the sequencing method may be a next generation sequencing technology.
  • next generation sequencing, or “NGS”, refers to a variety of sequencing techniques that permit simultaneous sequencing of millions of nucleic acid sequences, and is otherwise referred to as high-through put sequencing or massively parallel sequencing. Suitable NGS technologies are reviewed in, for example, Zhong et al., Ann Lab Med.
  • Suitable NGS technologies include, for example, second generation sequencing technologies such as pyro sequencing, ion torrent sequencing, and bridge PCR-based amplification methods.
  • second generation sequencing technologies such as pyro sequencing, ion torrent sequencing, and bridge PCR-based amplification methods.
  • pyrosequencing methods captures pyrophosphate (PPi) release and uses it as an indicator of specific base incorporation.
  • Ion torrent sequencing methods rely on hydrogen ion detection technology, which detects the release of protons during incorporation of nucleotides into the nucleic acid strand during synthesis.
  • Suitable bridge PCR- based amplification technologies include various Illumina platforms, such as MiSeq, MiniSeq, MiSeq, HiSeq, and NextSeq platforms.
  • Additional suitable NGS technologies include single molecule real-time (SMRT) technology, nanoball sequencing technology, and sequencing-by- binding technology, and scqucncing-by-hybridization technology.
  • sequencing comprises nanopore sequencing.
  • the method further comprises generating a methylation profile for the sample based upon said sequencing.
  • a “methylation profile” provides the methylation status of residues within the cut site flanking sequence.
  • a methylation profile provides a methylation status of CpG sites in the cut site flanking sequence.
  • a methylation profile identifies differentially methylated regions (DMRs). Identifying a methylation profile, methylation status, or differentially methylated region is inclusive of identifying/evaluating methylated and/or hydroxymethylated residues.
  • the methylation profile can provide valuable information about a disease state in a subject from which the sample was obtained.
  • the methylation profile can be used to classify tumors, including central nervous system (CNS) tumors, in a sample obtained from a subject.
  • DNA methylation is associated with cancer, autoimmune disease, metabolic disorders, and neurological disorders.
  • the methylation profile can be used to diagnose or classify cancer, autoimmune disease, a metabolic disorder, or a neurological disorder in a subject from which a sample was obtained.
  • the method generates a methylation profile of at least 100,000 loci. In some embodiments, the method generates a methylation profile of at least 1,000,000 loci.
  • the methods provided herein find use in classifying tumors in a subject from which a sample was obtained.
  • the methods provided herein are particularly advantageous over existing methods, in that the methods can be used in samples with low tumor fraction, fragmented DNA, and to classify as opposed to merely diagnose cancer in a subject.
  • the methods provided herein are thus particularly useful for early monitoring of tumor and selection of appropriate tumor treatment based upon the classification of the tumor.
  • the methods can be used to classify hard-to-diagnose tumors (e.g. brain tumors, sarcoma, undifferentiated tumors, and spindle tumors), atypical presentations, carcinoma of unknown primary, and from small or liquid biopsies thus without requiring surgical means to obtain a tissue sample.
  • the sample is a liquid biopsy sample (e.g. blood, serum, plasma urine, cerebrospinal fluid, or another biological fluid obtained from the subject containing tumor-derived entities such as circulating tumor cells, circulating tumor DNA, tumor extracellular vesicles, etc.).
  • the sample comprises cell free DNA (cfDNA).
  • the sample comprises less than lOOng DNA.
  • the sample comprises less than lOOng, less than 90ng, less than 80ng, less than 70ng, less than 60ng, less than 50ng, less than 40ng, less than 30ng, less than 25ng, less than 20ng, less than 15ng, or less than lOng DNA.
  • DNA methylation classification faces some challenges: 1) low and fragmented DNA input, as in the case of cell-free (cf) or degraded DNA from archived formalin-fixed paraffin-embedded (FFPE) samples, 2) low tumor fraction due to the high immune or stromal cell background, and 3) a minimal overlap (11%) between the currently mostly used probes derived from methylation arrays and the markers specific for cell types.
  • WGBS Whole genome bisulfite sequencing
  • FLEXseq Frametic Ligation Exclusive methylation sequencing
  • XRBS Extended-representation bisulfite sequencing
  • CCGG flanks Shareef, S. J. et al. Nat Biotechnol (2021) doi:10.1038/s41587-021-00910
  • FLEXseq provides superior results using samples such as fragmented and low-input DNA found in clinical specimens (e.g. cfDNA, FFPE tissue DNA).
  • a semi-permissive adapter was introduced that selectively blocks the free ends of non-target DNA, allowing FLEXseq to deliver a highly on-target and accurate methylome.
  • CCGG flanks cover 9 million CpGs with the highest yield closest to CCGG and 46% of CpGs in cell type-specific markers (Fig. 2a, Fig. 3a-c).
  • other methylation profiling methods including methylation microarrays, RRBS, and methylated DNA immunoprecipitation sequencing (MeDIP-Seq) only cover 3%-8% of those CpGs.
  • CCGG flanks also cover 36%-37% of CpGs from the machine learning (ME) classifiers based on methylation array data (central nervous system [CNS] or TCGA) (Fig. 2b).
  • CCGG flanks cover more enhancers, CpG shores/shelves, open sea, and introns than other methods (Fig. 3d).
  • FEEXseq was designed to target motifs containing a CpG site, output accurate methylation data, and be compatible with fragmented DNA input.
  • FEEXseq involves two ligations and a double-stranded cut between the ligations, followed by methylation conversion (Fig. la).
  • Adapter A semi-permissive adapter
  • a nuclease is then used to make double- stranded cuts, creating new and unblocked DNA ends.
  • Adapter B a second adapter that is also needed for sequencing library formation. Because each of the two adapters at opposite ends has a primer landing site (i.e. A+B are both needed), only targeted and cut DNA molecules are eventually sequenced while non-target DNA is ignored. Sequencing excludes non-target DNA with two A adapters or two B adapters. All DNA molecules have a free end associated with Adapter B that allows PCR duplicate removal and the potential of fragmentomics. The distinct free end is a major advantage compared to most profiling methods besides WGBS. Enzymatic conversion was then used instead of bisulfite conversion to avoid breaking the adapter-ligated molecules. Conversion is followed by amplification and sequencing of the target molecules.
  • the schematic shows targeting of a CCGG (SEQ ID NO: 1) motif.
  • a CCGG (SEQ ID NO: 1) is often referred to herein as targeted by FLEXseq
  • this CCGG (SEQ ID NO: 1) motif is only intended to be an exemplary motif containing a CpG site described herein, other motifs are also suitable.
  • the motif comprising a CpG site may comprise CCGG (SEQ ID NO: 1), TCGA (SEQ ID NO: 2), CGCG (SEQ ID NO: 3), CCGC (SEQ ID NO: 4), GCGC (SEQ ID NO: 5), or ACGT (SEQ ID NO: 6).
  • the nuclease used depends on the motif targeted.
  • the exemplary nuclease MspI is shown in FIG. 1 to target the CCGG (SEQ ID NO: 1), motif, but other suitable nucleases may be used to target other CpG containing motifs, as described herein.
  • the term “FLEXseq” does not necessarily indicate that the nuclease cuts at the CCGG cut site, but rather the nuclease may cut at any suitable motif containing a CpG site including any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.
  • an enzymatic conversion step is performed. However, as described herein, depending on the sequencing method used this enzymatic conversion step may be performed or not performed (e.g. for nanopore sequencing).
  • the term “FLEXseq” does not indicate that an enzymatic conversion step must be performed.
  • FIG. 4 A schematic of an exemplary process performed in accordance with the methods described herein, including exemplary sequences for adapters, primers, and target nucleic acids, is shown in FIG. 4.
  • FLEXseq had similar or higher correlations with WGBS than the public RRBS (Zhang, J. et al. Nat Commun 11, 3696 (2020) and XRBS (Shareef et al) data (Fig. 5c). These high correlations suggest that FLEXseq data is compatible with past datasets such as ones derived from the microarrays.
  • the median on-target rate of K562 DNA input titrations was 96% (IQR 95%- 96%) based on reads starting with (C/T)GG. Each on-target read is guaranteed to contain methylation data based on the first position. This high on-target rate yields an 18-, 6-, and 5-fold enrichment of CCGG flanks, cell type markers, and all CpGs compared with WGBS on a nucleotide basis (Fig. 2g). FLEXseq had the deepest coverage around the targeted CCGG cut sites (Fig. 2h, Fig. 3c).
  • FLEXseq provided chromosomal copy number output after normalizing against control diploid references from CSF cfDNA.
  • FLEXseq and whole genome sequencing (WGS) results were comparable with the exception of one case.
  • Tumor DNA was detected by interpreting deviations from diploid as CNAs and indicators of clonal aneuploidy. Tumor purity was estimated based on the difference between the log2 ratios of chromosomal segments and the baseline at gains and losses. Both measures were used in decision-making for the classifiers in the following sections.
  • DNA methylation profiling is being adopted clinically to adjudicate the diagnosis of CNS and solid tumors through tissue microarray analysis.
  • this approach was optimized to FLEXseq while reducing both DNA input requirements and cost.
  • a solid tumor (TCGA) ML classifier comprising 2,508 samples and 24,346 markers was developed (FIG. 6, FIG. 7, FIG. 8, and FIG. 9).
  • a parallel CNS tumor ML classifier consists of 2,801 samples and 16,429 markers (Fig. 10).
  • the estimated error rate of the ML calibrated scores was 10.2% for the TCGA and 1.0% for the CNS ML classifier.
  • the 45 tumor and control groups of the TCGA ML classifier are visualized using /-distributed stochastic neighbor embedding (t-SNE) dimensionality reduction (Fig. 6b).
  • Both ML classifiers were applied to DNA titrations of three tumor cell lines (breast invasive carcinoma [BRCA], colon carcinoma [COAD], and glioblastoma [GBM]) mixed into primary immune cell DNA background (B cells, T cells, monocytes, and neutrophils). Samples with higher tumor purities were accurately classified, while low-purity samples were classified with the control references (Fig. 6c).
  • BRCA breast invasive carcinoma
  • COAD colon carcinoma
  • GBM glioblastoma
  • CSF cfDNA was sequenced at a median of 154 million 2x50 reads (IQR 116-181 million).
  • the CCGG on-target rate remained high at 98%, similar to the initial K562 benchmark.
  • Fig. 17 show additional specimen characteristics and cfDNA inputs.
  • the TCGA and CNS microarray-based ML classifiers were used sequentially to maximize the coverage of different tumor entities. One of two misclassified cases (BF3027) and six of ten indeterminates were classified with their pathological diagnoses in the Z-SNE plots (Fig. 18a).
  • the deconvolution classifier (described above) had an overall accuracy of 96% (excluding seven indeterminates) and was 100% for LU AD, DLBC, and BRCA, respectively.
  • BF3683 was classified with STAD in the Z-SNE but was estimated to have a renal origin by deconvolution. Further charting showed that this patient had a concurrent renal cell carcinoma (RCC) not known to be in the CSF.
  • RCC renal cell carcinoma
  • BF3369 was also predicted to have a predominate renal origin, but the gold standard was also unclear based on unusual histology and non-matching clinical methylation classification testing of the tumor tissue
  • a composite tumor classifier integrating ML and deconvolution classification was developed (Fig. 16c).
  • the ML classifier was first used for its broad range of references, then deferred to the deconvolution classifier when cases had either low tumor purity ( ⁇ 50%, Fig. 18b), low classifier scores ( ⁇ 0.3), or classification as controls.
  • the deconvolution classifier improved the classification of low tumor purity and indeterminate cases that performed poorly in the ML classifier (Fig. 16d).
  • the composite classifier’s overall accuracy was 98% (excluding three indeterminates) and reached 100% accuracy for the three most prevalent tumor types: LUAD, DLBC, and BRCA (Fig. 16d-e).
  • FLEXseq a methylation enrichment profiling assay.
  • FLEXseq is demonstrated herein to have clinical relevance through copy number detection, ML classification, and deconvolution across different liquid biopsies and FFPE tissues. This method leverages both the breadth of references with ML classification and the lower tumor purity requirements with deconvolution classification. FLEXseq achieves an 18- fold enrichment covering CCGG flanking regions with an on-target rate of 96%-98%, guaranteeing methylation data from nearly every sequenced DNA molecule.
  • FLEXseq is highly concordant with the WGBS gold standard, accurately reflecting biological truth. Its genomewide coverage yields low-noise copy number plots. Its broad coverage allows for integration with past datasets and facilitates both ML and deconvolution classification with external references.
  • TET, BGT, and APOBEC enzymatic conversions were used herein, but alternatives include other non-destructive conversion strategies such as TAPS, DM-seq, or SEM- seq. Bisulfite conversion is possible, albeit with DNA loss. Because the enrichment occurs before PCR, FLEXseq is also compatible with direct methylation readout using nanopore sequencing. Moreover, the design of FLEXseq is not limited to the MspI nuclease, which is restricted to CCGG motifs. Other nucleases like CRISPR-Cas9 can target alternative flanking regions. For example, the Taql-v2 enzyme was used to cut at the CpG containing motif TCGA (SEQ ID NO: 2).
  • Results are shown in FIG. 19.
  • the on-target rate of using the TaqLv2 nuclease (NEB, pail number R0149) targeting the 'TCGA' motif rather than MspI. Cutsmart buffer was used.
  • the on-target rate was estimated by dividing reads starting with CGA or TGA by the total reads on the side cut by the nuclease. Each on-target read is guaranteed to contain CpG methylation data based on the first position.
  • WGBS is the gold standard, but its high sequencing and computational costs bar large-scale studies or clinical testing.
  • Methylation microarrays are commonly used but require 250 ng of DNA input and cover only 2%-4% of all CpGs and 3%-8% of cell type markers (Fig. 2a), limiting deconvolution accuracy. Building array reference sets is costly due to the need to scale to thousands of samples.
  • the EPICv2 array costs $265 per sample and $70 for FFPE repair. In this study, 160M reads amount to $114 per sample (NovaseqX 25B kit).
  • microarrays are restricted to human and mouse genomes.
  • RRBS is effective for CpG islands but covers fewer enhancers and cell type-specific markers (Fig. 3).
  • XRBS targets CCGG flanks like FEEXseq but was not designed for fragmented DNA found in clinical specimens.
  • MeDIP-seq targets methylated cytosines but imprecisely detects hypomethylated markers, encompassing 98% of cell type-specific markers.
  • FEEXseq As a cost-efficient yet broad profiling technology, FEEXseq opens future possibilities. By preserving one free end on every DNA molecule, it has the potential to be utilized in fragmentomics. FEEXseq captures half of the known methylation aging markers associated with epigenetic clocks and is poised to broaden the identification of aging markers. FLEXseq can be used to explore the methylome in non-human organisms. Genotypes and mutations can be phased with the methylation status in over 95% of reads. Finally, this data is compatible with metagenomics, which could potentially provide information relevant to infectious diseases.
  • FLEXseq represents a clinical tool for low-input and low-purity samples from small and/or liquid biopsies. As a research tool, it offers base-pair resolution of the methylome, providing an economical yet comprehensive alternative to whole genome sequencing and microarrays.

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Abstract

La présente divulgation concerne des procédés d'analyse d'acides nucléiques dans un échantillon. En particulier, la divulgation concerne des procédés d'analyse d'un échantillon impliquant l'enrichissement d'acides nucléiques contenant des sites CpG dans un échantillon et l'évaluation de l'état de méthylation dans des acides nucléiques enrichis.
PCT/US2024/056111 2023-11-16 2024-11-15 Classification d'échantillons à l'aide d'un enrichissement de méthylation Pending WO2025106807A1 (fr)

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Citations (4)

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Publication number Priority date Publication date Assignee Title
US20090047680A1 (en) * 2007-08-15 2009-02-19 Si Lok Methods and compositions for high-throughput bisulphite dna-sequencing and utilities
US20210277459A1 (en) * 2007-02-07 2021-09-09 Illumina Cambridge Limited Preparation of templates for methylation analysis
US20230130140A1 (en) * 2020-09-30 2023-04-27 Guardant Health, Inc. Methods and systems to improve the signal to noise ratio of dna methylation partitioning assays
US20230235320A1 (en) * 2020-06-24 2023-07-27 Claret Bioscience, Llc Methods and compositions for analyzing nucleic acid

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210277459A1 (en) * 2007-02-07 2021-09-09 Illumina Cambridge Limited Preparation of templates for methylation analysis
US20090047680A1 (en) * 2007-08-15 2009-02-19 Si Lok Methods and compositions for high-throughput bisulphite dna-sequencing and utilities
US20230235320A1 (en) * 2020-06-24 2023-07-27 Claret Bioscience, Llc Methods and compositions for analyzing nucleic acid
US20230130140A1 (en) * 2020-09-30 2023-04-27 Guardant Health, Inc. Methods and systems to improve the signal to noise ratio of dna methylation partitioning assays

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